Team:Lethbridge/Notebook

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October

October 1

Roxanne Gel for gel extraction

Restriction Ribo-Chez-dT

  • 2x CheZ-dT
  • 2x GFP-dT
  • 3x C-EYFP-dT

Ligation

  • C-ECFP-dT
  • C-CFP-dT
  • C-EYFP-dT
  • mRBS-N-ECFP
  • mRBS-N-EYFP
  • pSB-C-EFP
  • pSB-C-EYFP
  • pSB-C-CFP
  • pSB-N-ECFP
  • pSB-N-EYFP

Aliquoted DH5α cells

Ashley

Tube Tube + Gel Gel
1.1226g 1.4931g 0.2671g
1.012g 1.2302g 0.1290g

Eluted with 50uL buffer EB

Ligation

1:3 2:6
lum-dT 3uL 6uL
SRBS 1uL 2uL
Ligase Buffer 1uL 2uL
Water 4.5uL 9uL
Ligase 0.5uL 1uL

Oct 2

Pick Colonies:

  • sRBS + Lumazine-dT
  • mRBS + N-term FPs
  • C-term FPs + dT
  • pSB + N-term FPs
  • pSB + N-term FPs

Gel Extraction:

  • Riboswitch-cheZ-dT
  • 2x cheZ-dT (in case Ribo-cheZ-dT isn't actually there)
  • 3x C-EYFP-dT
  • 2x GFP-dT

Liquid Culture:

  • Riboswitch-cheZ-dT
  • 2x cheZ-dT (in case Ribo-cheZ-dT isn't actually there)
  • 3x C-EYFP-dT
  • 2x GFP-dT

Miniprep:

  • Mr. Gene mms6

Sat Oct 3

Miniprep:

  • sRBS + Lumazine-dT
  • mRBS + N-term FPs
  • C-term FPs + dT
  • pSB + N-term FPs
  • pSB + N-term FPs
  • sRBS + Lumazine-dT

Restriction:

Analytic and Prep:

  • mRBS + N-term FPs
  • C-term FPs + dT
  • pSB + N-term FPs
  • pSB + N-term FPs

Analytic only:

  • Mr. Gene mms6

Prep only:

  • pBAD-TetR

Oct 5

Gel extraction:

  • sRBS + Lumazine-dT
  • mRBS + N-term FPs
  • C-term FPs + dT
  • pSB + N-term FPs
  • pSB + N-term FPs

Ligation:

  • pStrong + Riboswitch-cheZ-dT
  • Riboswitch-GFP-dT
  • pLac + sRBS-Lumazine-dT
  • pBad-TetR + C-term FPs
  • pBad-TetR + N-term FPs
  • pTetR + N-term

Transformation:

  • pLac + sRBS-Lumazine-dT
  • pBad-TetR + C-term FPs
  • pBad-TetR + N-term FPs
  • pTetR + N-term ECFP
  • pStrong + Riboswitch-cheZ-dT
  • Riboswitch-GFP-dT

Sequencing:

  • sRBS + Lumazine-dT
  • mRBS + N-term FPs
  • C-term FPs + dT
  • pSB + N-term FPs
  • pSB + N-term FPs
  • Mr. Gene mms6

Tues Oct 6

Pick Colonies:

  • pLac + sRBS-Lumazine-dT
  • pBad-TetR + C-term FPs
  • pBad-TetR + N-term FPs
  • pTetR + N-term ECFP
  • pStrong + Riboswitch-cheZ-dT
  • Riboswitch-GFP-dT

Wed Oct 7

Miniprep:

  • pLac + sRBS-Lumazine-dT
  • pBad-TetR + C-term FPs
  • pBad-TetR + N-term FPs
  • pTetR + C-term ECFP
  • pTetR + N-term ECFP
  • pStrong + Riboswitch-cheZ-dT
  • Riboswitch-GFP-dT

Analytic Restriction:

  • pLac + sRBS-Lumazine-dT
  • pBad-TetR + C-term FPs
  • pBad-TetR + N-term FPs
  • pTetR + N-term ECFP
  • pStrong + Riboswitch-cheZ-dT
  • Riboswitch-GFP-dT

Prep Restriction:

  • Riboswitch-GFP-dT

Thurs Oct 8

Gel Extraction:

  • Riboswitch-GFP-dT

Ligation:

  • pStrong + Riboswitch-GFP-dT

Transformation:

  • pStrong + Riboswitch-GFP-dT

Sequencing:

  • pLac-sRBS-Lumazine-dT
  • pBad-TetR-C-term FPs
  • pBad-TetR-N-term FPs
  • pTetR-N-term ECFP
  • pStrong-Riboswitch-cheZ-dT
  • Riboswitch-GFP-dT

Maxiprep:

  • pLac-sRBS-Lumazine-dT
  • pBad-TetR-C-term FPs
  • pBad-TetR-N-term FPs
  • pTetR-N-term ECFP
  • pStrong-Riboswitch-GFP-dT

Restriction:

  • pLac-sRBS-Lumazine-

Analytic and Prep:

  • pBad-TetR-C-term FPs
  • pBad-TetR-N-term FPs
  • pTetR-N-term ECFP
  • C-term CFP (has pTetR)

Analytic Only:

  • pStrong-Riboswitch-GFP-dT

Fri Oct 9 Overexpression pLac + sRBS-Lumazine-dT Gel Extraction pLac-sRBS-Lumazine-dT pBad-TetR-C-term FPs pBad-TetR-N-term FPs pTetR-N-term ECFP C-term CFP (has pTetR) Motility Media

Oct 12

Ligation:

  • pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
  • pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Transformation:

  • pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
  • pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Plate Testing:

  • pStrong-Riboswitch-GFP-dT

Motility Test:

  • pStrong + Riboswitch-cheZ-dT

Oct 13

Pick Colonies

  • pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
  • pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Analysis of Plate and Motility Tests

Wed Oct 14

Miniprep:

  • pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
  • pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Restriction:

  • pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
  • pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Thurs Oct 15

Gel Extraction:

  • pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
  • pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Ligation:

  • pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
  • pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Transformation:

  • pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
  • pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Expression Testing:

  • pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
  • pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Fri Oct 16

Pick Colonies:

  • pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
  • pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Sat Oct 17

Maxiprep:

  • pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
  • pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

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